Tar DNA-Binding Protein, 43 kDa, (TDP-43) is a multifunctional DNA/RNA binding protein. TDP-43 Proteinopathy is a heterogeneous spectrum of neurodegenerative disorders that includes cases of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). Like other neurodegenerative disorders, TDP-43 proteinopathy represents a substantial societal burden, and yet lacks early diagnostic tools or disease-modifying therapies. Evidence supports the idea that either mutations in or dysregulation of TDP-43 lead to TDP-43 proteinopathy. However, the biological function of TDP-43 and the molecular mechanisms by which TDP-43 leads to TDP-43 proteinopathy remain to be elucidated. One facet that has not been fully explored is the role of microRNAs. MicroRNAs (i.e. miRNAs) are a class of non-coding RNAs that regulate mRNA targets by binding them and destabilizing them. Each miRNA can target dozens of mRNAs, and each mRNA can be targeted by multiple miRNAs. Thus, a complex network of miRNA-mRNA interactions emerges in any cell. Though it is known that TDP-43 regulates miRNA biogenesis, which miRNAs are regulated, what their biological function is, and whether they have a role in the pathogenesis of TDP-43 proteinopathy have not been systematically characterized. To address these questions, this proposal has two aims. In the first aim, miRNA expression profiles will be collected from mouse cortical neurons to determine which miRNAs change after TDP-43 knock-down. Then, paired expression profiles of miRNAs and mRNAs will be collected in this knock-down model, as well as in a cellular mouse model of TDP-43 proteinopathy that the lab has developed, and in post-mortem brain tissue samples from TDP-43 proteinopathy patients. With these paired expression profiles, a bioinformatics approach will then be used to first predict what miRNA-mRNA interactions are occurring. It will then predict which particular miRNAs and mRNAs are having the largest impact on four functional network domains (TDP-43 aggregation, neuronal survival, axonal integrity, and mitochondrial function) dysregulated in TDP-43 proteinopathy. In the second aim, experiments will address whether modulating the interactions of three predicted miRNAs and three target genes will impact the four functional domains. The lab has recently developed a novel microfluidics chamber with a coupled algorithm, which will be utilized to assess axonal integrity in an unbiased and efficient manner. The lab has also established cell biological, imaging and biochemical assays to study the other functional domains. The insights gleaned from these two aims will contribute to a foundation of knowledge about the biological function of TDP-43 and the pathogenesis of TDP- 43 proteinopathy that will help develop early diagnostic tools and disease-modifying therapies.